While appropriate mechanical loading, such as exercise, is beneficial for maintaining tendon homeostasis, chronic, repetitive mechanical loading may lead to the development of tendinopathy, which is the leading cause of chronic disabilities that affects millions of people just in the United States alone. The typical histopathological features of tendinopathy have been identified, including accumulation of lipid cells, mucoid degeneration, and tissue calcification, either alone or in combination. These findings suggest the presence of cells with multi-phenotypes that differ from residential tenocytes, which express the fibroblast phenotype. During the course of investigation for our parent project, we have identified tendon stem cells, which constitute a sub-population of tendon cells and can undergo adipogenic, chondrogenic, and osteogenic differentiation. Therefore, we are now in good position to introduce stem cell biology into our investigation of the pathogenic mechanisms for the development of tendinopathy. Furthermore, accumulating evidence has pointed to the prominent role of adult stem cells in tissue pathologies, such as tumorigenesis. Thus, in this study, we aim to test a novel hypothesis that tendon stem cells play a major role in the development of tendinopathy by differentiating in "wrong" non-tenocyte directions (e.g., adipocytes, chondrocytes, and osteocytes) in response to chronic, repetitive mechanical loading placed on tendons. Therefore, the specific aim of this project is: To determine the effect of chronic, repetitive mechanical loading via treadmill running on the differentiation of mouse tendon stem cells (MTSCs) We will use histochemistry and immunohistochemistry to detect changes in the tendon matrix and cell phenotype in tendon sections. We will also extract tendon cells from tendon samples, characterize the differentiation state of MTSCs, and determine phenotypes of differentiated MTSCs (tenocytes and non- tenocytes) by gene and protein expression analysis (RT-PCR and Western blot), flow cytometry (i.e. FACS) analysis, and immunocytochemistry. Finally, we will use a novel biophysical approach, cell traction force microscopy (CTFM), to assess MTSC differentiation state and verify the results of biochemical analysis. This study is a logical extension of our parent project and is the first to investigate the role of tendon stem cells in the development of tendinopathy. The findings of this study will bring new insights into the precise pathogenesis of tendinopathy and may lead to the development of novel therapies for slowing down or reversing tendon degeneration, a hallmark of tendinopathy at later stages. [unreadable] [unreadable] [unreadable]